1. Field of the Invention
This invention relates in general to certain new and useful improvements in biological determination reagent composition which permits the stabilization of enzymes and coenzymes in an aqueous media therein, and the method of stabilizing, and, more particularly, to such reagent composition and methods useful in determination of creatine phosphokinase.
2. Description of the Prior Art
It has recently been estimated that 25% of all in vitro diagnostic tests conducted annually in this country are not reliable. Unreliable tests can result in unnecessary medical treatment, the withholding of necessary treatment and lost income. Because of their high specificity, the use of enzyme determinations has significantly increased during the last few years, and indications are that this trend will continue. However, rigorous quality control measures are required to assure the accuracy and consistency of results. This requirement stems from the fact that the exact nature of enzymes, as well as the mechanisms of their action, remains unknown for the most part.
At present, the greatest limitation on the enzyme reagent manufacturer, by far, lies in the unstable characteristics of his products. Current methodologies require the use of numerous labile ingredients, and these ingredients are more likely to increase, rather than decrease, in number. Due to these severe restraints, rigorous quality control is required, and this quality control is, of course, costly. Moreover, if control in any step in the process is not maintained within high degree of control standards, the quality of the final product can be reduced materially.
The present commercial state of the art used for stabilizing the reactive ability of enzymes or coenzymes is by locking them into a solid matrix, either by freeze drying, dry blending such as used for tableting dried powders, primarily in the pharmaceutical diagnostic and related industries and immobilization by locking the chemical structure of the enzyme into a solid matrix. Contrary to the sophistication these terms imply, these approaches are neither practical nor desirable and are also expensive. The manufacturer is forced to remove the water and supply a partial product, thus relinquishing part of the quality control cycle in the dilution and use of the final product. Laboratories are forced to pay the high cost of packaging, reagent waste, freeze drying and dry blending, and usefulness of the product is further limited by packaging modes and sizes.
Furthermore, good product uniformity is difficult to achieve. This condition is exemplified by the fact that most commercial freeze dried control sera (reference serum) lists the acceptable bottle-to-bottle variation of enzyme constituents at .+-.10% of the mean.
The present invention is uniquely designed so that the labile ingredients in a diagnostic reagent composition are effectively stabilized. The composition comprises three separate solutions and the first of which is a substrate solution which contains a labile substrate and at least one labile enzyme. The second solution is a coenzyme solution which contains at least one labile coenzyme and may contain a labile enzyme. The third solution is a buffer solution which includes a buffer agent to maintin the desired pH when the three solutions are mixed at time of use. The labile components are effectively stabilized in aqueous solutions against reactivity, and the means of stabilization ensures long-term stability in a liquid media. Moreover, close tolerance control can be achieved in the manufacturing of a high quality product which eliminates the inconvenience of the rigid package size and the high cost of packaging and freeze drying and reagent waste.